Compounds for enzyme amplification assay

ABSTRACT

Novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification. By employing receptors specific for one or a group of materials (hereinafter referred to as &#39;&#39;&#39;&#39;ligands&#39;&#39;&#39;&#39;) and binding an enzyme to the ligand or ligand counterfeit to provide an &#39;&#39;&#39;&#39;enzyme-bound-ligand,&#39;&#39;&#39;&#39; an extremely sensitive method is provided for assaying for ligands. The receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efficiencies of enzyme-boundligand and enzyme-bound-ligand combined with receptor. The receptor, ligand and enzyme-bound-ligand are combined in an arbitrary order and the effect of the presence of ligand on enzymatic activity determined. Various protocols may be used for assaying for enzymatic activity and relating the result to the amount of ligand present.

United States Patent [191 Rubenstein et al.

[ Dec. 3, 1974 1 COMPOUNDS FOR ENZYME AMPLIFICATION ASSAY [75]Inventors: Kenneth E. Rubenstein, Palo Alto;

Edwin F. Ullman, Atherton, both of Calif.

[73] Assignee: Syva Corporation, Palo Alto, Calif.

[22] Filed: Nov. 6, 1972 [21] Appl. No.: 304,157

Related US. Application Data [63] Continuation-impart of Ser No 143,609,May 14,

1971, abandoned.

[56] References Cited UNITED STATES PATENTS 2/1972 Csizmas et a1. 195/634/1972 Schuurs et a1. 195/1035 R Primary ExaminerAlvin E. TannenholtzAttorney, Agent, or FirmTownsend and Townsend 5 7 ABSTRACT Novelbiological assay method for determining the presence of a specificorganic material by employing a modified enzyme for amplification. Byemploying receptors specific for one or a group of materials(hereinafter referred to as ligands) and binding an enzyme to the ligandor ligand counterfeit to provide an enzyme-bound-ligand, an extremelysensitive method is provided for assaying for ligands. The receptor whenbound to the enzyme-bound-ligand substantially inhibits enzymaticactivity, providing for different catalytic efficiencies ofenzyme-bound-ligand and enzyme-bound-ligand combined with receptor. Thereceptor, ligand and enzyme-bound-ligand are combined in an arbitraryorder and the effect of the presence of ligand on enzymatic activitydetermined. Various protocols may be used for assaying for enzymaticactivity and relating the result to the amount of ligand present.

11 Claims, N0 Drawings COMPOUNDS FOR ENZYME AMPLIFICATION ASSAY CROSSREFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of Application Ser. No. 143,609, filed May 14, 1971and now abandoned.

BACKGROUND OF THE INVENTION 1. Field of the Invention There is acontinually pressing need for rapid, accurate qualitative andquantitative determinations of biologically active substances atextremely low concentrations. The purpose of the determination can beextremely varied. Today, there is a wide need for determining thepresence of drugs or narcotics in body fluids, such as saliva, bloord orurine. In addition, in medical diagnosis, it is frequently important toknow the presence of various substances which are synthesized naturallyby the body or ingested. These include hormones, both steroidal andpolypeptides, prostaglandins, toxines, as well as other materials whichmay be involved in body functions. Frequently, one is concerned withextremely small amounts and occassionally, with very small differencesin concentrations.

To meet these needs, a number of ways have been devised for analyzingfor trace amounts of materials. A common method is to use thin layerchromatography (TLC). By determining the flow factors and using specificreagents, the presence of certain materials can be detected; in manyinstances the particular material can be isolated and identifiedquantitatively, for example, by mass spectroscopy or gas phasechromatography However, thin layer chromatography has a number ofdeficiencies in being slow, requiring 'a high degree of proficiency inits being carried out, being subject to a wide range of interferingmaterials, and suffering from severe fluctuations in reliability.Therefore, the absence of satisfactory alternatives has resulted inintensive research efforts to determine improve methods of separationand identification.

An alternative to thin layer chromatography has been radioimmunoassay.Here, antibodies are employed for specific haptens or antigens. Aradioactive analog employing a radioactive atom of high flux is used andbound to the antigen. By mixing an antibody with solutions of the haptenor antigen and the radioactive hapten or antigen analog, the radioactiveanalog will be prevented from binding to the antibody in an amountdirectly related to the concentration of the hapten or antigen in thesolution. By then separating the free radioactive analog from theantibody bound radioactive analog and determining the radioactivity ofthe separate components, one can determine the amount of hapten orantigen in the original solution.

The use of radioactive materials is not desirable for a variety ofreasons. First, radioactivity creates handling problems and undesirablehazards. Secondly, the preparation of such compounds involves similarhazards, greatly enhanced by the much larger amounts of radioactivematerials which are present. Because of their instability, theradioactive materials have only a short life. In addition, the use ofradioactive materials requires a license from the Atomic EnergyCommission, subjecting the licensee to review by the Commission as tothe maintenance of minimum operating standards. These standards maychange from time to time, so as to involve added expense andinconvenience to the licensee. Finally, the separation of the bound andunbound radioactive analog is difficult and subject to error. See, forexample, Abraham, Prelim. Comm., 29, 866 (1969).

Besides the aforementioned materials, assays at extremely lowconcentrations would be desirable for a variety of pesticides, such asinsecticides, bactericides, fungicides, etc., as well as other organicpollutants, both in the air and water. Organic pollutants may be assayedwhenever a receptor can be devised and the pollutant is inert to thereagents employed.

2. Description of the Prior Art Use of radioimmunoassay is described intwo articles by Murphy, 1. Clin. Endocr. 27, 973 (I967); ibid., 28, 343(I968). The use of peroxidase as a marker in an immunochemicaldetermination of anitgens and antibodies is found in Stanislawski etal., C. R. Acad. Sci. Ser. D. 1970, 271 (16), 1442-5. (CA. 74 1144 B).See also, Nakane, et al., J. of Histochem. and Cytochem. 14, 929 (1967)and Avrameas, Int. Rev. of Cytology, 27, 349 (1970). A generaldescription of thin layer chromatography for assay may be found inStahl, Thin Layer Chromatography, Springer Verlag, New York, 1969. Seealso, Peron, et al., Immunologic Methods in Steroid Determination,Appleton, Century'Crofts, New York, 1970.

Also of interest are publications by Van Weemen, et al., FEBS Letters14, 232 (1971), and Engvall, et al., Immunochemistry, 8, 871 (I971)concerned with immunoassays employing enzymes. See also US. Pat. No.3,654,090. See also, Cinader, Proceedings of the Second Meeting of theFoundation of European Biochemical Societies, Pergamon, Oxford, 1967,vol. II, chapter four.

SUMMARY OF THE INVENTION Detection of ligands is obtained at extremelylow concentrations by using specific receptor sites for the ligand andenzyme applification of ligand displacement. By bonding a ligand or aligand counterfeit to an enzyme while retaining enzymatic activity andthen combining the enzyme-bound-ligand to a receptor for the ligand, thepresence and amount of ligand in an unknown solution may be readilydetermined. By competition for receptor sites between theenzyme-boundligand and the free ligand, the two ligand moieties beingadded to the receptor simultaneously or sequentially, the difference inenzymatic activity resulting from the presence or absence of ligand maybe determined in accordance with a particular analytical scheme. Thisdifference will be related to the amount of ligand present in theunknown solution. Enzymatic activity is easily determined in known waysby following the change in concentration of an enzyme substrate orproduct of the substrate by standard techniques.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS This invention provides a methodfor detecting or assaying extremely low concentrations of a wide rangeof organic materials by relating the presence of a particular unknown toenzymatic activity. An amplification is obtained by having a largenumber of molecules formed or transformed as a result of the presence ofone molecule. This amplification is achieved by bonding the compound tobe assayed or a counterfiet of the compound to an enzyme. Thisassemblage is referred to as an enzyme-bound-ligand. The particularmolecule to be assayed is referred to as a ligand. The ligand analogwill include either a ligand which is modified by replacing a protonwith a linking group to bond to the enzyme or a ligand counterfeit whichis a ligand modified by other than simple replacement of a proton toprovide a linking site the enzyme. The ligand and the enzymebound-ligandare both capable of binding in a competitive fashion to specificreceptor sites. It should also be noted that other compounds of verysimilar structure may serve as ligands capable of competing for thesesites, e.g., morphine glucuronide and codeine will compete withenzyme-bound-morphine for binding to certain types of morphineantibodies. In most instances, this is advantageous in permitting one toassay for a class of physiologically closely related compounds.

Various methods'or protocols may be employed in assaying for a widevariety of ligands. Normally, the ligand, enzyme-bound-ligand andreceptor will be soluble in the medium employed. The substrate(s) forthe enzyme may or may not be soluble in the medium. In some situationsit may be desirable to provide a synthetic substrate which is notsoluble or employ an insoluble natural substrate.

in carrying out the assay,-the enzyme-bound-ligand is combined with ahigh molecular weight receptor which results in inhibition ofenzymaticactivity, When a ligand and enzyme-bound-ligand are introducedinto a solution containing ligand receptor, the enzymatic activity ofthe solution after the three substances are com.- bined will be affectedby the concentration of the ligand present in the solution. That is, theenzymebound-ligand and the ligand will compete for the receptor sites.The number of enzymebound-ligand molecules not inhibited by the receptorwill be directly related to the number of ligand molecules present inthe solution. One can achieve this in two ways: (I) either bycompetition, whereby the enzyme-bound-ligand and ligand are introducedto the receptor substantially simultaneously; or (2) theenzyme-bound-ligand or ligand may be first added to the receptor, andthe system allowed to come to equilibrium, and then the ligand added orenzyme-bound-ligand added respectively, in effect, to displace thematerial originally added from the receptor. Since the enzymaticactivity will be diminished or inhibited when the enzyme-bound-ligand isbound to the receptor, the enzymatic activity of the solution will bedirectly related to the amount of ligand present in the solution.

The assay can be carried out, either by considering the effect of ligandon the rate at which enzyme-boundligand binds to receptor or the effectof ligand on the equilibrium between the reagents: enzyme-boundligandand receptor. Where enzyme-bound-ligand and ligand are presentwith'receptor, one need not wait until equilibrium is achieved betweenthe three species. If one measures the enzymatic acitivity at a specifictime or interval of time from the time of combination of the threespecies, the enzymatic activity of the assay mixture will be a functionof the effect of the ligand on the rate of binding of theenzyme-bound-ligand to the receptor. By determining standards under thesame conditions, including the same time interval, employing differentconcentrations of ligand, a smooth standard curve is obtained.

By measuring the effect of the ligand on rate of binding, rather thanthe effect of equilibrium, a shorter time interval between the time ofcombining the reagents and unknown suspected of containing the ligandand the time for the determination will be involved, compared withwaiting until equilibrium is achieved. it is frequently found thatreproducible values can be obtained in from 0.1 to 5 minutes aftercombining the reagents and unknown. The rate of enzymatic activity isusually determined over a short time interval, e.g.. l minute. The timeinterval can be the second, third, etc. minute from the time when thereagents and unknown were combined.

The concentrations of the reagents: the enzymebound-ligand and thereceptor, may be varied widely. Normally, the concentration of receptor(based on active sites) and enzyme-bound-ligand will be from about 10 toIO M, more usually from 10* to 10 M. The lower limit for theconcentration of enzymebound-ligand is predicated onthe minimum amountwhich can be detected. This will vary with different enzymes as well asdifferent detection systems.

The amount of receptor employed is normally calculated based on receptorsites and will vary with the concentration of enzyme-bound-ligand, theratio of ligand to enzyme in the enzyme-bound-ligand, and the affinityof the receptor for the ligand. Usually, there will be at least 1 activereceptor site per molecule of enzymebound-ligand and less than about 20active sites per molecule of ligand as enzyme-bound-ligand, butsiteligand molecule ratios may be as high as l,000 to 1, depending onthe type of assay and the affinity of the receptor. Preferably, theratio of receptor active sites to molecules of enzyme-bound-ligand willbe at least one, usually at least two, and the ratio of active sites tomolecules of ligand as enzyme-bound-ligand will be less than about 5 tol. The ratio will vary to a great degree depending on binding constantsand the amount of ligand suspected of being present. The method ofdetermining binding sites for the receptor will be discussedsubsequently in the experimental section.

The en'zyme-bound-ligand will usually have mole cules of ligand toenzyme subunit ratios on the average over the entire composition in therange of 0.01 :1, frequently 0.0250:l, and more frequently about 0.0425:1, wherein the number of ligands when the ligand is a protein isexpressed as the number of ligand molecules times the number of itscomponent polypeptide chains. For small ligands (less than about 10,000molecular weight), there will generally be at least one ligand, moreusually at least two ligands per enzyme, while with large ligands(greater than about 5,000 molecular weight) there will generally be atleast one enzyme per ligand. In the area of overlap, the ratio willdepend on the nature of the ligand, among other factors to be discussed.

, The number of small ligands per enzyme will be affected to some degreeby the molecular weight of the enzyme. However, normally, the fewermolecules ofgand bound to an enzyme to achieve the desired degree ofinhibitability with receptor, the more sensitive the assay. Therefore,the number of small ligands per enzyme will usually not exceed 40, moreusually not exceed 30, and will not exceed 1 ligand per 2,000 molecularweight of enzyme on the average over the entire composition. Usually,the range of ligands will be 1 to 40, more usually I to 24, and withrandom substitution 2 to 20.

With large ligands, there will be on the average not more than oneenzyme per 2,000 molecular weight, usually not more than one enzyme per4,000 molecular weight, and more usually not more than one enzyme per6,000 molecular weight.

In some instances, a number of enzymes bind together in a stablearrangement to form a multienzyme complex. Because of the juxtapositionof the enzymes, a number of reactions may be carried out sequentially inan efficient manner, providing localized high concentrations ofreactions. Therefore, the ligand may be bound to a combination ofenzymes, whereby there will be a plurality of enzymes per ligand. If anumber of ligands were bound to the multienzyme complex, one could have1:1 mole ratio of enzymes to ligand, although, in fact, there would be aplurality of enzymes and ligands involved in a single aggregation. Thenumberof enzymes bound together, either as a multienzyme complex or byanother mechanism will rarely exceed 20, usually not exceed 10, andcommonly be in the range of 2 to 5 enzymes.

All other things being equal, the greater the number of enzymes perlarge ligand, the greater the sensitivity of the assay. However, theenzymes may interfere with receptor recognition, affect solubility andbe deleterious in other ways. Therefore, usually, the number of enzymesbonded to a large ligand will be such that there will be no more thanone enzyme polypeptide chain for every 2,000 molecular weight of theligand.

The concentration of receptor and enzyme will be related to the range ofconcentration of the ligand to be assayed. The solution to be assayedwill be used directly, unless a relatively high concentration of ligandis present. If a high concentration is present, the unknown solutionwill be diluted so as to provide a convenient concentration. However, inmany biological systems of interest, the amount of material beingassayed will be relatively small and dilution of the unknown substratewill usually not be required.

To illustrate the subject method, a soluble receptor is employed for aparticular ligand. For illustrative purposes, the ligand will beconsidered the hapten, morphine, and the receptor will be an antibodyspecific for morphine. It should be notedparenthetically, thatantibodies generally recognize molecular shape and distribution of polargroups in a ligand, although a portion of the ligand may besignificantly modified without preventing recognition. For example, bothmorphine and its glucuronide can be bound to certain morphineantibodies.

An enzyme is first modified by bonding one or more morphine molecules tothe enzyme; a sufficient number of morphine groups are employed so thatgreater than about percent inhibition, usually 50 percent inhibition,and preferably, at least 70 percent inhibition is obtained when themaximum number of ligands are conjugated to receptor. Completeinhibition is usually neither necessary or desirable. In many instances,all that is required is that there be a measurable difference betweencompletely uninhibited and maximally inhibited enzyme-bound-ligand whichwould allow for a semiquantitative or quantitative determination of aligand through a desired range of concentrations. Any convenient enzymecan be used that will catalyze the reaction of a substrate that can beeasily detected and for which a substrate is available which allows forinhibition of the enzyme when bound to receptor.

A solution is prepared of the antibody at the requisite concentration.Only a few microliters of solution are required. The antibody,maintained at a pH at which it is active in binding morphine, isintroduced into a solution of the enzyme-bound-morphine at the desiredconcentration. The reactivity of the combined antibody andenzyme-bound-morphine solution can be determined by taking an aliquot,adding it to its substrate under conditions where the enzyme is active,and determining the spectroscopic change as a function of time at aconstant temperature. The rate of this change will be the result thatshould be obtained when there is no morphine present in the unknownsolution.

Normally, the ligand and enzyme-bound-ligand reversably bind toreceptor, so that the order of addition of reagents is not crucial.

A second aliquot is taken and added to the unknown solution. The unknownsolution may contain the substrate and any other additives which arerequired for enzymatic activity. Alternatively, the unknown solution mayfirst be combined with the antibody-(enzymebound-morphine) complex,allowed to come to equilibrium and then mixed with the substrate' Ineither case the rate of change in the spectrum is determined. A variantof the above method is to add combined enzyme-bound-morphine and unknownsolution to the antibody and then add this solution to the substrate.Other obvious variations come readily to mind.

If all concentrations of reagents except morphine are kept constant andseveral standard solutions of morphine are employed, then one can relatethe change in the spectrum over specified periods of time to themorphine concentrations. Obviously, the standardized sys tem can then beused to determine rpaidly, accurately, and efficiently the amount ofmorphine, or any other ligand in the unknown.

The manner of assaying for the enzyme can be widely varied depending onthe enzyme, and to some degree the ligand and the medium in which theligand is obtained. Conveniently, spectrophotometric measurements can beemployed, where absorption of a cofactor, a substrate or the product ofthe substrate absorbs light in the ultraviolet or visible region.However, in many instances other methods of determination may bepreferred. Such methods include fluorimetry, measuring luminescence, ionspecific electrodes, viscometry, electron spin resonance spectrometry,and metering pH, to name a few of the more popular methods.

The assays will normally be carried out at moderate temperatures,usually in the range of from 10 to 50 C, and more usually. in the rangeof about 15 to 40 C. The pH of the assay solutions will be in the rangeof about 5 to 10 usually about 6 to 9. Illustrative buffers include(trishydroxymethyl)-methylamine salt, carbonate, borate and phosphate.

Whether oxygen is present or the assay is carried out in an inertatmosphere, will depend on the particular assay. Where oxygen may be aninterferant, an inert atmosphere will normally be employedpNormally,hydroxylic media will be employed, particularly aqueous media, sincethese are the media in which the enzyme is active. However 0 to 40 vol.percent of other liquids may also be present as co-solvents, such asalcohols, esters, ketones, amides, etc. The particular choice of thecosolvent will depend on the other reagents present in the medium, theeffect on enzyme activity, and any desirable or undesirable interactionswith the substrate or products.

As already indicated, antibodies will frequently recognize a family ofcompounds, where the geometry and spatial distribution of polargroupsare similar. Frequently, by devising the haptenic structure and themethod of binding to the antigen when producing the antibodies, thespecificity of the antibody can be varied. In some instances, it may bedesirable to use two or more antibodies, usually not more than sixanitbodies, so that the antibody reagent solution will be able to detectan entire group of compounds, e.g., morphine and barbiturates. This canbe particularly valuable for screening a sample to determine thepresence of any member of a group of compounds or determining whether aparticular class of compounds is present, e.g., drugs of abuse or sexhormones. When combinations of antibodies are used, it will usually benecessary to employ corresponding combinations of enzymepounds for whichreceptors can be provided range from simple phenylalkylamines, e.g.,amphetamine, to very high molecular weight polymers, e.g., proteins.

Among ligands which are drugs, will be compounds which act as narcotics,hypnotics, sedatives, analgesics, antipyretics, anaesthetics,psychotogenic drugs, muscle relaxant's, nervous system stimulants,anthcholinesterase agents, parasympathomimetic agents, sympathomimeticagents, a-adrenergic blocking agents, antiadrenergic agents, ganglionicstimulating and blocking agents, neuromuscular agents, histamines,antihista mines, S-hydroxy-tryptamine and antagonists, cardiovasculardrugs, anitarrhythmic drugs, antihypertensive agents, vasodilator drugs,diuretics, pesticides (fungicides, antihelminthics, insecticides,ectoparasiticides, etc.), antimalarial drugs, antibioticsantimetabolites, hormones, vitamins, sugars thyroid and antithyroiddrugs, corticosteroids, insulin, oral hypoglemic drugs, tumor cells,bacterial and viral proteins, toxins, blood proteins, and theirmetabolites.

(A drug is any chemical agent that affects living protoplasm. (Goodmanand Gilman, The Pharmacological Basis of Therapeutics, 3rd Ed.,Macmillan, New York (1965).) A narcotic is any agent that produces sleepas well as analgesia.)

included among such drugs and agents are alkaloids, steroids,polypeptides and proteins, prostaglandins, catecholamines, xanthines,arylalkylamines, heterocyclics, e.g., thiazines, piperazines, indoles,and thiazoles, amino acids, etc.

Other ligands of interest besides drugs are industrial pollutants,flavoring agents, food additives, e.g., preservatives, and foodcontaminants. v

Broadly, the ligands will be organic compounds of from 100 to 100,000molecular weight, usually of from about 125 to 40,000 molecular weight,more usually 125 to 20,000 molecular weight. The ligand will usuallyhave from about 8 to 5,000 carbon atoms and from about 1 to 3,500heteroatoms.

A substantial portion of the ligands will be monomers or low orderpolymers, which will have molecular weights in the range of about to2,000 more usually to 1,000. Another significant portion of the ligandswill be polymers (compounds having a recurring group) which will havemolecular weightsin the range of from about 750 to 100,000, usually fromabout 2,000 to 60,000, more usually 2,000 to 50,000. For polymers ofvarying molecular weight, weight average molecular weight is intended.

In some instances, high molecular weight materials will be of interest.For example, blood proteins will generally be in excess of 100,000molecular weight. In the case of lipoproteins, the molecular weight willbe in the range of 3 million to 20 million. The globulins, albumins andfibrinogens will be in the range of 100,000 to 1,000,000.

The ligands will normally be composed of carbon, hydrogen, nitrogen,oxygen, sulfur. phosphorous, halogen, and metals, primarily as theircations, such as the alkali and alkaline earth metals and the metals ofGroups 18, H8, V118, and VlllB, particularly the third row of theperiodic chart. Most usually, the ligands will be composed primarily ofcarbon, hydrogen, nitrogen, oxygen and sulfur.

Structurally, the ligands may be monomers or polymers, acyclic, mono orpolycyclic, having carbocyclic or heterocyclic rings. The ligands willhave a wide variety of functionalities, such as halo, oxocarbonyl,nonoxocarbonyl, amino, oxy (hydroxy, aryloxy, alyloxy and cycloalyloxy[alyl intends a monovalent aliphatic radicall), thiooxy, dithio,hydrazo, and combinations thereof.

The ligands may be divided into three different categories, based ontheir biological relationship to the receptor. The first category isantigens, which when introduced into the bloodstream of a vertebrate,result in the formation of antibodies. The second category is haptens,which when bound to an antigenic carrier, and the hapten bound antigeniccarrier is introduced into the bloodstream of a vertebrate, elicitformation of antibodies specific for the hapten. The third categoryofligands includes those which have naturally occuring receptors in aliving organism and the receptors can be isolated in a form specific forthe ligand.

Of course, biological substances which are native to one species andhave naturally occurring receptors in that species, may also be haptenswhen bonded to a protein and a introduced into an animal of the same ora different species. Therefore, the classification is somewhat arbitraryin that the ligand may be an antigen as to one species, a hapten as toanother species, and may have naturally occurring receptors in a thirdspecies.

Anitgens are for the most part protein or polysaccharide in nature andforeign to the animal into which they are injected.

The most important body of ligands for the purposes of the invention arethe haptens. Substances which on injection do not give rise toantibodies, but which are able to react with antibodies specifically toproduce either precipitation or to inhibit precipitation have beentermed haptens. This definition has been used to include not only thesimple chemical substances which are determinants of specificity whenconjugated to protein, and which inhibit precipitation, but alsosubstances obtained from natural sources such as the pneumococcal typespecific polysaccharides and dextran which are not antigenic in therabbit on primary injection." Kabat, et al., ExperimentalImmunochemistry, Charles C. Thomas, Springfield, Illinois (1967). In thefollowing discussion the term hapten will be confined to groupsartificially introduced into antigenic carriers which promote theformation of antibodies to those groups.

The third group of ligands are those which have naturally occurringreceptors. The receptors may be proteins, nucleic acids, such asribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or membranesassociated with cells. Illustrative ligands which have naturallyoccurring receptors are thyroxine, many steroids, such as the estrogens,cortisone, corticosterone, and estradiol; polypeptides such as insulinand angiotensin, as well as other naturally occurring biologicallyactive compounds. See Murphy, et al., J. Clin. Endocr., 24, 187 (1964);Murphy, ibid, 27, 973, (I967); ibid, 28, 343 (I968); BBA, 176, 626,(1969); McEwen, et al., Nature, 226, 263 (1970); Morgan, et al.,Diabetes, (1966); Page, et al., J. Clin. Endocr., 28, 200, (1969).

The ligands may also be categorized by the chemical families which havebecome accepted in the literature. In some cases, included in the familyfor the purpose of this invention, will be those physiominmeticsubstances physiomimetic are similar in structure to a part of thenaturally occurring structure and either mimic or in- .hibit thephysiological properties of the natural substances. Also, groups ofsynthetic substances will be included, such as the barbiturates andamphetamines. In addition, any of these compounds may be modified forlinking to the enzyme at a site that may cause all biological activityto be destroyed. Other structural modifications may be made for the easeof synthesis or control of the characteristics of the antibody. Thesemodified compounds are referred to as ligand counterfeits.

A general category of ligands of particular interest are drugs andchemically altered compounds, as well as the metabolites of suchcompounds. The interest in assaying for drugs varies widely, fromdetermining whether individuals have been taking a specific illicitdrug, or have such drug in their possession, to determining what drughas been administered or the concentration of the drug in a specificbiological fluid.

The drugs are normally of from eight carbon atoms to 40 carbon atoms,usually of from nine to 26 carbon atoms, and from 1 to 25, usually fromone to 10 heteroatoms, usually oxygen, nitrogen or sulfur. A largecategory of drugs have from one to two nitrogen atoms.

One class of drugs has the following basic functionalwhere the linesintend a bond to a carbon atom, and wherein any of the carbon atoms andthe nitrogen atom may be bonded to hydrogen, carbon or aheterofunctionality. Drugs which have this basic structure include theopiates such as morphine and heroin, meperidine, and methadone.

Another class of drugs are the epinephrine like drugs which have thefollowing basic functionality;

where the lineintend a bond to a carbon atom and wherein any of thecarbon atoms and the nitrogen atom may be bonded to hydrogen, carbon ora heterofunctionality. Drugs which have this basic structure includeamphetamine, narceine, epinephrine, ephedrine and L-dopa.

The ligand analogs of drugs will usually have molecular weight in therange of to 1,200 more usually in the range of to 700.

Alkaloids The first category is the alkaloids. Included in the categoryof alkaloids, for the purpose of this invention, are those compoundswhich are synthetically prepared to physiologically simulatethenaturally occurring alkaloids. All of the naturally occurringalkaloids have an amine nitrogen as a heteroannular member. Thesynthetic alkaloids will normally have a tertiary amine, which may ormay not be a heteroannular member. The alkaloids have a variety offunctionalities present on the molecule, such as ethers, hydroxyls,esters, acetals, amines, isoxazole, olefins, all of which, depending ontheir particular position in the molecule, can be used as sites forbonding to the enzyme.

Opiates The opiates are morphine alkaloids. All of these molecules havethe following functionality and minimum structures:

wherein the free valences are satisfied by a wide variety of groups,primarily carbon and hydrogen.

The enxyme-bound-ligand analog of these compounds will for the most parthave the following mini-.

mum skeletal Stl'UClIUI'BI wherein X is a bond or a functionality suchas imino, azo, oxy, thio, sulfonyl, oxocarbonyl, nonoxocarbonyl, orcombinations thereof. Oxygen will be in the ortho, meta or B position. Ais an enzyme which is bonded to X at other than its reactive site andretains a substantial portion of its natural enzymatic activity. Therewill be m ligands bonded through X to the enzyme A.

The enzyme-bound-morphine and its closely related analogs will have thefollowing formula:

wherein: i

any one of the W groups can be X* or an H of any i of the W groups maybe replaced by X*, wherein X* is a bond or a linking group;

A* is an enzyme bonded at other than its reactive site, having a number(n) of ligands in the range of 1 to the molecular weight of A* dividedby 2,000, usually in the range of 2 to 40;

W is hydrogen or hydrocarbon of from one to eight carbon atoms,particularly alkyl or alkenyl of from one to four carbon atoms,cycloalkylalkyl of from four to six carbon atoms, or aralkyl, e.g.,methyl, allyl, 3-methylbut-2-enyl-l, cyclopropylmethyl and ,8-phenethyl;

W is hydrogen; W is hydrogen;

W is hydrogen or taken together with W a divalent radical of from threeto six carbon atoms and to 2 oxygen atoms, forming a six memberedcarbocyclic ring with the carbon chain to which they are attached, e.g.,propylene-l ,3, l -hydroxyprop-2-enylenel ,3, l hydroxypropylenel ,3,l-acetoxypropylenel ,3, l acetoxyprop-2-enylene-l ,3 l -oxopropylene-l ,3l oxoprop-2-enylene-l ,3;

W is hydrogen or hydroxyl;

W is hydrogen, hydroxyl or taken together with W y W is hydrogen ormethyl;

W is hydrogen, methyl or hydroxyl;

W is hydrogen, hydroxy, acyloxy of from one to three carbonatorns, e.g.,acetoxy, (unless otherwise indicated, acyl intends only nonoxocarbonyl),hydrocarbyloxy of from I to 3 carbon atoms, e.g., methoxy, ethoxy,2-(N-morpholino)ethoxy and glucuronyl; and

W is hydrogen. (It is understood that in all the formulas, except when aminimum or skeletal structure is indicated, unsatisfied valences aresatisfied by hydrogen).

(Hydrocarbyl is an organic radical composed solely of hydrogen andcarbon and may be saturated or unsat- W oxy (-0-); and

W is hydroxy, acetoxy, or alkoxy of from one to three carbon atoms;

Those preferred compounds having the basic morphine sturcture will havethe following formula:

O J i N-W\ l\/ i 1 i l 7 wherein: one of W" and W is X**; when otherthan X**; W" is methyl; and W is hydrogen, methyl, acetyl or glucuronyl;W is hydrogen or acetyl, usually hydrogen;

-X** is wherein Z is hydrocarbylene of from one to seven carbon atoms,preferably aliphatic, having from 0 to I site of ethylenic unsaturation;and

-Z** is an enzyme, either specifically labelled with n equal to l to 2ligands or randomly (random as to one or more particular availablereactive functionalities) labelled with n equal to 2 to 30, more usually2 to 20, the enzyme retaining a substantial proportion of its activity.The enzyme will be of from about 10,000 to 300,000, frequently about10,000 to l50,000 molecular weight and is preferably an oxidoreductase,e.g., malate dehydrogenase, lactate dehydrogenase, glyoxylate reductase,or glucose 6-phosphate dehydrogenase, or a glycosidase, e.g., lysozymeor amylase.

Illustrative opiates which can be bound to an enzyme include morphine,heroin, hydromorphone, oxymorphone, metopon, codeine, hydrocodone,dihydrocodeine, dihydrohydroxycodeinone, pholcodine, dextromethorphan,phenazocine, and dionin and their metabolites.

Preferred compounds have W, or W as X*-A* or have W and W taken togetherto provide A*X- *CHCH CH or A*X*CH-CH=CH. Methadone Another group ofcompounds having narcotic activity is methadone and its analogs, whichfor the most part have the following formula:

wherein:

any one of the W groups can be X*; X*, A*, and'n have been definedpreviously; p is or 1, usually being the same in both instances;

q is 2 or 3;

W is hydrogen;

W and W are hydrogen, alkyl of from one to three carbon atoms, e.g.,methyl, or may be taken together to form a six-membered ring with thenitrogen atom to which they are attached, e.g., pentylene-l,5 and 3-oxaor 3-azapentylene-l,5;

W is hydrogen or methyl, only one W being methyl;

W is hydrogen; W is hydrogen or hydroxyl; W is hydrogen, acyloxy of fromone to three carbon atoms, e.g., propionoxy, or hydroxy (when W and Ware both hydroxy, the oxo group is intended); and

W" is hydrogen or alkyl of from one to three carbon atoms, e.g., ethyl.

Illustrative compounds which can be linked to an enzyme are methadone,dextromoramide, dipipanone, phenadoxone, propoxyphene (Darvon) andacetylmethadol.

Metabolites of methadone and methadone analogs are also included. Amongthe metabolites for methadone is N-methyl 2-ethyl 3,3-diphenyl-5-methylpyrroline.

Preferred compounds are when W or W is -X*.

A narrower class of methadone and its analogs are of 55 the formula:

wherein: 7 any one of the W groups can be X*;

. gen atom and the carbon atom to which W"" X*, A* and n have beendefined previously;

W and W' are hydrogen;

W' and W are methyl or are taken together with the nitrogen atom towhich they are attached to form a morpholino or piperidine ring;

W and W*' are hydrogen, hydroxy, acetoxy, at least one being hydroxy oracetoxy; and

W is alkyl of from one to three carbon atoms.

, The methadone derivatives will for the most part have the followingformula:

w 'o-owncn,onmormw H 1 I A wherein:

one of W" or W" is X**; X**, A**, and n have been defined previously;(1) is phenyl; I

when other than X** W" is methyl; and

W"" is propyl.

The metabolites of methadone and close analogs will for the most parthave the following formula:

wherein:

any one of the W groups can be X*, X*, A* and n have been definedpreviously;

is hydrogen,. hydroxyl, methoxyl or acetoxyl, that is of from one to twocarbon atoms, and except when hydrogen of from one to two oxygen atoms;

W is hydrogen, methyl, or a free valence joined with W W is an unsharedpair of electrons;

W is hydrogen or methyl; I

W is hydrogen, hydroxy, or taken together with W""' forms a double bondbetween the nitroand W are respectively attached; and

W""' is alkyl of from one to three carbon atoms, usually two carbonatoms, or maybe taken together with W to form alkylidenyl of from one tothree carbon atoms, usually two carbon atoms.

Preferred compounds are those where W or W""' are X*, particularly Wwith W"' as methyl. I

phenylbenzyl( l- The third group of compounds which have narcoticactivity and are meperidine or meperidine analogs, have for the mostpart the following formula:

wherein:

any one of the W groups can be X*;

X*, A*, and n have been defined previously;

W is hydrogen;

W is hydrogen, alkyl of from one to three carbon atoms, e.g., methyl,aminophenylalkyl, e.g., B-(p aminophenyl)ethyl, or phenylaminoalkyl,e.g., phenylaminopropyl, (alkyl of from two to three carbon atoms);

W is alkoxy of from one to three carbon atoms, e. g., ethoxy; and

W is hydrogen or methyl.

Illustrative compounds are meperidine, alphaprodine, alvodine andanileridine.

Preferred compounds are those where W or W is X* or a hydrogen of W isreplaced with X*. indole Alkaloids A second group of ligands of interestare based on tryptamine and come within the class of indole alkaloids,more specifically ergot alkaloids. These compounds will have thefollowing minimal structure:

wherein the free valences are satisfied by a variety of groups,primarily carbon and hydrogen, although other substituents may bepresent such as carboxyl groups, hydroxyl groups, keto groups, etc. Themost common member of this class which finds use is lysergic acid,primarily as its diethylamide. Other members of the indole alkaloidfamily which can also be assayed for are the strychnine group and theindolopyridocoline group, which finds yohimbine and reserpine asmembers.

The enzyme substituted indole alkaloids will have the following formula:

wherein m, X and A have been defined previously.

Other groups of alkaloids include the steroid alkaloids, the iminazolylalkaloids, the quinazoline alkaloids, the isoquinoline alkaloids. thequinoline alkaloids, quinine being the most common, and the diterpenealkaloids.

For the most part, the alkaloids bonded to an enzyme will be of fromabout 300 to 1,500 molecular weight, more usually of from about 400 to1,000 molecular weight. They are normally solely composed of carbon,hydrogen, oxygen, and nitrogen; the oxygen is present as oxy and 0x0 andthe nitrogen present as amino or amido. Catecholamines The first groupin this category are Catecholamines of the formula:

wzm xv i w w W was I o-w '37 wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen or alkyl of from one to three carbon atoms, e.g., methyl;

W is hydrogen, or alkyl of from one to three carbon 5 atoms, e.g.,methyl;

Illustrative compounds include cotainine, narceine,

. noscapine and papaverine.

Preferred compounds are where W W or W are X* or have a hydrogenreplaced with X*.

A group of compounds related to the Catecholamines are epinephrine,amphetamines and related compounds. These compounds have the formula:

wherein: any one of the W groups can be X*;

X*, A* and n have been defined previously;

W and W are hydrogen or alkyl of from one to three carbon atoms, e.g.,methyl and isopropyl, preferably one is hydrogen;

W is hydrogen, alkyl of from one to three carbon atoms, e.g., methyl andethyl, or may be taken together with W to form a ring having six annularmembers with the nitrogen as the only heteroatom;

W is hydrogen, hydroxyl, carbomethoxy, or may be taken together with W"to form a morpholine ring;

W is carbomethoxy, when W and W are taken together to form a pip'eridinering; and

W and W are hydrogen, hydroxyl or alkoxyl of from one to three carbonatoms.

Illustrative compounds which can be bonded to an enzyme are ephedrine,epinephrine, L-dopa, benzidrine (amphetamine), paredrine,methamphetamine, methyl phenidate and norephedrine.

Illustrative compounds which can be linked to an enzyme include3-(3,4'-dihydroxyphenyl)-3-hydroxypropionic acid,N-(B-(B,3,4-trihydroxyphen)ethyl) N- methyl glycine,N-(l-phenyl-2-propyl)oxalamic acid, l-phenyl-2-methylamino-l-propyl)glycolic acid, p-

(2-methylaminopropyl-l )phenoxyacetic acid, N-( l '-phenyl-2-propyl)glycine, 4-methylamino-4-phenylvaleric acid, para-( 2- aminopropyl-l)phenoxyacetic acid,

4-methylamino-5-phenylvaleric acid, and 3-amino-4- phenylbutyric acid.

Where W and W are hydrogen, preferred compounds will have the followingformula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W' and W are hydrogen or alkyl of from one to three carbon atoms,preferably one is hydrogen;

W is hydrogen, methyl or may be taken together with W' to form apiperidine ring;

W is hydrogen, hydroxyl or carbomethoxy; and

W is hydrogen.

Where W and W" are oxy, the preferred compounds have the followingformula:

W43" is hydrogen or hydroxyl; and ndcfliila e rhydwayl rr rncthq y WClosely related compounds to the amphetamines are those where asaturated five or six membered ring is substituted for the phenyl ring.These compounds will have the following formula:

wherein:

any oneof the W groups is X*; X*, A* and n have been defined previously;

W' have been defined above; W' is hydrogen or methyl; W' is hydrogen orhydroxyl;

W is hydrogen; and

thyl)barbituric b is an integer of from four to five. Of particularinterest are those amphetamines bonded to enzymes of the followingformula:

W is hydrogen;

W is methyl; and

W"' is hydrogen;

W is hydrogen or methyl;

X** is ZCO, wherein Z is hydrocarbylene of from one to seven carbonatoms, usually aliphatic, having from 0 to I site of ethylenicunsaturation, with the proviso that when W"' is -X**, X** is O-ZCO-;

A** and n have been defined previously. Barbiturates A wide class ofsynthetic drugs which finds extensive and frequent abuse are thebarbiturates. These compounds are synthetically readily accessible andtheir use only difficultly policed. The compounds which find use willcome within the following formula:

wherein:

any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W is hydrogen, alkyl of from one to three carbon atoms, e.g., methyl oralkali metal, e.g., sodium;

W and W are hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, or arylhydrocarbon of from one to eight, more usually one to six carbon atoms,e.g., ethyl, nbutyl, a-methylbutyl, isoamyl, allyl, A -cyclohexenyl, andphenyl;

W is hydrogen, or alkali metal, e.g., sodium;

W is oxygen or sulfur.

Illustrative compounds are veronal, medinal, luminal, prominal, soneryl,nembutal, amytal, dial, phenadorn. seconal, evipan, phenobarbital andpentothal.

Preferred compounds would have W or W or a hydrogen of W or W as --X*.Also preferred is when one of W and W is hydrocarbyl of from two toeight carbon atoms.

Illustrative compounds which may be linked to an enzyme include5,5-diethyl-l-carboxymethylbarbituric acid,S-ethyl-S-n-butyl-l-succinoylbarbituric acid, 5- ethyl-S-phenyll N 2'-chloroethyl )-2 '-aminoeacid, 5-(2'-carboxy-A" cyclohexenyl )-l,S-dimethylbarbituric acid, carboxymethyl phenobarbital, S-(y-crotonicacid)-5- (2'-pentyl)-barbituric acid, 5-(p-aminophenyl)-5-ethylbarbituric acid, 5-(5'-pentanoic acid) -5-(2'-pen- 19tyl)barbituric acid, and l-methyl--ethyl-5-(p-carboxyphenyl)barbituricacid.

Of particular interest are those barbiturates bonded to an enzyme of theformula:

wherein one of W and W is X**; when other than X**:

W is hydrogen, methyl or alkali metal, e.g., sodie; and

W is hydrocarbon of from one to eight carbon atoms, having from 0 to 1site of ethylenic unsaturation;

W is hydrocarbon of from two to eight carbon atoms, having from O to 1site of ethylenic unsaturation;

X** is ZCO, wherein Z is hydrocarbylene of from one to seven carbonatoms, usually aliphatic, having from O to 1 site of ethylenicunsaturation;

A** and n have been defined previously.

Glutethimide Another compound of interest is glutethimide, wherein theenzyme bound analog will have the following formula: 3 0

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W and W are hydrogen; and

W is lower alkyl of from one to three carbon atoms, e.g., ethyl. CocaineA drug of significant importance in its amount of use is cocaine. Theenzyme bound cocaine or cocaine metabolites or analogs, such asecgonine, will for the most part have the followingformula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydroxy, methoxy, amino or methylamino;

W is hydrogen or benzoyl; and 60 W is hydrogen or alkyl of from one tothree carbon atoms, e.g., methyl.

Of particular interest are those ecgonine derivatives (including cocainederivatives) of the formula:

wherein one of W and W when other than X**:

W is hydrogen or benzoyl; and W is methyl;

W is hydroxy or methoxy; X** is wherein Z" is methylene or carbonyl; or

Z-CO wherein Z is hydrocarbylene of from one to seven carbon atoms,usually aliphatic, having from 0 to I site of ethylenic unsaturation;

A** and n have been defined previously. Diphenyl Hydantoin Anothercompound of interest is the antiepileptic drug diphenyl hydantoin. Thiscompound and its analogs will have the following formula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

()5 is phenyl;

W W" and W are hydrogen. Marijuana Because of its ready availability andwidespread use, tetrahydrocannabinol (the active ingredient of marijuana) and its congeners, cannabidiol and cannabinol and theirmetabolites are compounds of great interest, where a simple assay methodwould be of importance. The compounds which find use as analogs have thefollowing formula:

Wino

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W( is hydrogen or carboxyl;

W" is hydroxyl or methoxyl;

W" is hydrogen;

W is pentyl or hydroxypentyl;

W" is hydrogen, methyl, or the two W s may be taken together to form acarbocyclic ring of from 5 to 6 annular members; and

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W" and W are amino.

The next group of tranquilizers are benzdiazocycloheptanes and are knownas Librium, Valium, Diazepam, or Oxazepam. These compounds and theirrelated analogs will have the following formula:

wherein:

any one of the W groups can be -X*; X*, A*, and n have been definedpreviously; W' and W are hydrogen;

W is hydrogen, lower alkyl of from one to three carbon atoms, e.g.,methyl, or may be taken together with W to form a double bond betweenthe carbon andthe nitrogen;

W is amino or lower alkylamino of from one to three carbon atoms, e.g.,methylamino, or may be taken together with W" to form a carbonyl;

W" is hydrogen or hydroxyl; and

W" is oxy or an unshared pair of electrons.

The next group of compounds are the phenothiazines of whichchlorpromazine is a member. These compounds will for the most part havethe following forwherein:

any one of the W groups can be X*;

X*, A*, and n have been defined previously;

W" is hydrogen, alkyl of from one to six carbon atoms, dialkylaminoalkylof from four to eight carbon atoms, e.g., 3-(dimethylamino)propyl;N-hydroxyalkyl (alkyl of from two to three carbon atoms), N'-piperazinoalkyl (alkyl of from two to three carbon atoms), e.g.,N-hydroxyethyl N'-piperazinopropyl; N-

'alkyl (alkyl of from one to three carbon atoms) N'- piperazinoalkyl(alkyl of from two to three carbon atoms), e.g., N-methylN-piperazinopropyl; and Z-(N- alkyl)-piperidinoalkyl, wherein theN-alkyl is of from one to three carbon atoms and the other alkyl is offrom two to three carbon atoms, e.g., 2-(N-methyl)- piperidinoethyl,there being at least two carbon atoms between the heteroatoms;

W is hydrogen, chloro, trifluoromethyl, alkylmercapto of from one tothree carbon atoms, e.g., methylmercapto and acyl of from one to threecarbon atoms, e.g., acetyl; and

W" and W are hydrogen.

Amino Acids, Polypeptides and Proteins The next group of compounds arethe amino acids, polypeptides and proteins. For the most part, the aminoacids range in carbon content from two to 15 carbon atoms, and include avariety of functional groups such as mercapto, dithio, hydroxyl, amino,guanidyl, pyrrolidinyl, indolyl, imidazolyl, methylthio, iodo,diphenylether, hydroxyphenyl, etc. These, of course, primarily primarilthe amino acids related to humans, there being other amino acids foundin plants'and animals.

Polypeptides usually encompass from about 2 to amino acid units (usuallyless than about 12,000 molecular weight). Larger polypeptides arearbitrarily called proteins. Proteins are usually composed of from 1 to20 polypeptide chains, called subunits, which are associated by covalentor non-covalent bonds. Subunits are normally of from about I00 to 400amino acid groups (-l0,000 to 50,000 molecular weight).

Individual polypeptides and protein subunits will normally have fromabout-2 to 400, more usually from about 2 to 300 recurring amino acidgroups. Usually, the polypeptides and protein subunits of interest willbe not more than about 50,000 molecular weight and greater than about750 molecular weight. Any of the amino acids may be used in preparingthe polypeptide.

Because of the wide variety of functional groups which are present inthe amino acids and frequently present in the various naturallyoccurring polypeptides, the enzyme bonded compound can be bonded to anyconvenient functionality. Usually, the enzyme bonded compound can bebonded to a cysteine, lysine or arginine, tyrosine or histidine group,although serine, threonine, or any other amino acid with a convenientfunctionality, e.g., carboxy and hydroxy, may be used.

For the most part, the enzyme-labeled polypeptides will have thefollowing formula:

ine, histidine, methionine, hydroxyproline, tryptophan,

tyrosine, thyroxine, omithine, phenylalanine, arginine, and lysine.Polypeptides of interest are ACTH, oxytocin, lutenizing hromone,insulin. Hence-Jones protein, chorionic gonadotropin, pituitarygonadotropin, growth honnone, rennin, thyroxine bonding globulin,

bradykinin, angiotensin, follicle stimulating hormone, etc.

' In certain instances, it will be desirable to digest a protein andassay for the small polypeptide fragments. The concentration of thefragment may then be related to the amount of the original protein.

Steroids Another important group of compounds which find use in thisinvention are the steroids, which have a wide range of functionalitiesdepending on their function in the body. In addition to the steroids,are the steroidmimetic substances, which while not having the basicpolycyclic structure of the steroid, do provide some of the samephysiological effects.

The steroids have been extensively studied and derivatives preparedwhich have been bonded to antigenic proteins for the preparation ofantibodies to the steroids. Illustrative compounds include:l7B-estradiol-6- (O-carboxymethyl-oxime)-BSA (bovine serum albumin)(Exley, et al., Steroids 18 593, (1971 testosterone-3-oxime derivativeof BSA (Midgley, et al., Acta Endocr. 64 supplement 147, 320 (1970));and progesterone-3-oxime derivative of BSA (Midgley, et al., ibid.)

For the most part, the steroids used have the following formula:

wherein m, X and A have been defined previously. Usually, the enzymewill be bonded to the A, B, or C rings, at the 2, 3, 4, 6 or 11positions, or at the 16 or 17 positions of the D ring or on the sidechains at the 17 position. Of particular interest is where X is bondedto the 6 position. The rings may have various substituents, particularlymethyl groups, hydroxyl groups, oxocarbonyl groups, ether groups, andamino groups. Any of these groups may be used to bond the enzyme to thebasic ring structure. For the most part, the steroids of interest willhave at least one, usually l to 6, more usually I to 4 oxygenfunctionalities, e.g., alcohol, ether, esters, or keto. In addition,halo substituents may be present. The steroids will usually have from 18to 27 carbon atoms, or as a glycoside up to 50 carbon atoms.

The rings may have one or more sites of unsaturation, either ethylenicor aromatic and may be substituted at positions such as the 6, 7 and l 1positions with oxygen substituents. In addition, there may be methylgroups at the and 13 positions. The position marked with a Z, 17, may beand will be varied widely depending on the particular steroid. Zrepresents two monovalent groups or one divalent group and may be acarbonyl oxygen,an hydroxy group, an aliphatic group of from one toeight carbon atoms, including an acetyl group, an hydroxyacetyl group,carboxy or carboxyalkyl of from two to six carbon atoms, an acetylenicgroup of from two to six carbon atoms or halo substituted alkyl oroxygenated alkyl group or a group having more than one functionality,usually from I to 3 functionalities.

For the second valence of Z, there may be a H or a second group,particularly hydroxyl, alkyl, e.g., methyl, hydroxyalkyl, e.g.,hydroxymethyl; halo, e.g., fluoro or chloro, oxyether; and the like.

These steroids find use as hormones, male and female (sex) hormones,which may be divided into oestrogens, gestogens, antrogens,adrenocortical hormones (gluco corticoids), bile acids, cardiotonicglycosides and aglycones, as well as saponins sapogenins.

Steroid mimetic sutstances, particularly sex hormones are illustrated bydiethyl stilbestrol.

The sex hormones of interest may be divided into two groups; the malehormones (androgens) and the female hormones (oestrogens).

The androgens which find use will have the following formula:

\I AW...

W m w, y

we: m

0-1 site of cthylenic uusnturution wherein: I

any one of the W groups can be -X*; X*, A* and n have been definedpreviously; W is hydrogen, or hydroxyl; W is hydrogen, methyl orhydroxy] (when two groups bonded to the same carbon atom are hydroxyl,

oxo is intended);

W and W are hydrogen or hydroxyl, at least one of W is hydroxy (eitheras hydroxy or oxo);

W is hy drogen or two W 5 may be taken together to form a double bond;

W is methyl; and

W is hydrogen.

Illustrative compounds which may be bonded to an enzyme includetestosterone, androsterone, isoandrosterone, etiocholanolone,methyltestosterone and de hydroisoandrosterone.

Illustrative compounds which may be linked to an enzyme includeN-carboxymethoxy testosteroneimine, l7-monotestosteronyl carbonate,androsteronyl succinate, testosteronyl maleate, O -carboxymethyl 0'-methyl androst-S-ene-BB, l7B-diol, testosterone O-carboxypropyl oximeand androsteronyl carbonate.

The oestrogens have an aromatic A ring and for the most part have thefollowing formula:

wr2 W WG-1 site of etliylcnic unsaturation W and W" are hydrogen,ethinyl, or hydroxyl (when two hydroxyls are bonded to the same carbonatom, oxo is intended);

W is hydrogen or hydroxyl;

W is hydroxyl or alkoyl of from one to three carbon atoms;

W is hydrogen or two W s may be taken together to form a double bond;and

W is hydrogen.

Illustrative compounds which may be bonded to an enzyme are equilenin,B-estradiol, estrone, estriol, and

l7-a-ethinyl-estradiol.

Illustrative compounds which may be linked to an enzyme include3-carboxymethyl estradiol, 2- chloromethylestrone, estrone glutarate,Ocarboxymethyloxime of 6-ketoestradiol, equilenyl N- carboxymethylthiocarbamate.

Another class of hormones are the gestogens which have the followingformula:

wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W and W are hydrogen or hydroxyl, at least one being hydroxyl (where twohydroxyl groups are bonded to the same carbon atom, oxo is intended);

W is hydrogen or hydroxyl;

W and W are hydrogen or being hydroxyl; and

W is hydrogen, or two W s may be taken together to form a double bond.

Illustrative compounds which may be bonded to an hydroxyl, at least oneenzyme include progesterone, pregnenolone, allopregwas Q-l site ofthylonic unsatumtion wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W is hydrogen or hydroxyl;

W and W are hydrogen or hydroxyl, at least one of which is hydroxyl(when two hydroxyl groups are bonded to the same carbon atom, oxo isintended);

W is hydrogen or hydroxyl;

W W W and W are hydrogen or hydroxyl, at

least one of W and W is hydroxyl;

W is methyl or formyl; and

W is hydrogen or two W s may be taken together to form a double bond.

Illustrative compounds which may be bonded to an enzyme are17-hydroxydioxycorticosterone (Compound S), deoxycorticosterone,cortisone, corticosterone, l l-dihydrocortisone (Compound F), cortisol,prednisolone and aldosterone.

Illustrative compounds which may be linked to an enzyme include O-carbox ymethyl corticosterone, N- carboxymethyl 2l-carbamate cortisol,2 l-cortisone succinate, 2l-deoxocorticosterone succinate, and 0"-methyl, O -carboxymethyl cortisone-i An additional steroid family is thecardiotonic glycosides and aglycones of which digitalis is an importantmember. The basic compound is digitoxigenin, which is also found as theglycoside. The compounds of interest have the following formula:

0-1 site of cthylenic unsaturation CO 5 l l wit-z wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously;

W", W, W" and W are hydrogen, hydroxyl, or a glycoside, at least onebeing hydroxyl or a sugar, mostly as a glycoside. The sugars includexylose, glucose, cymarose, rhamnose, and galactose.

Also of interest are the saponins and sapogenins derived from plants.These compounds have a spiro ring structure at C Vitamins Sugars Thenext group of compounds are the sugars and saccharides. The saccharidesare combinations of various -l site of ethylenic unsaturatian wherein:

any one of the W groups can be X*;

X*, A* and n have been defined previously; W is hydrogen or hydroxyl;

W and. W are hydrogen or hydroxyl (where two hydroxyl groups are bondedto the same carbon atom,

oxo is intended);

W is hydrogen or hydroxyl; and

W is hydroxyl, amino or an oxy group of from one to six carbon atoms,e.g., alkoxy.

Miscellaneous Included in this group are the antibiotics such aspenicillin, chloromycetin, antinomycetin, tetracycline, terramycin, andnucleic acids or derivatives, such as nucleosides and nucleotides.

Also of interest is serotonin which is 3-(2- aminoethyl)5hydroxyindole.X* may be bonded at either of the amino nitrogen atoms or the hydroxylgroup. 7

Of course, many of the compounds which are of interest undergo metabolicchanges, when introduced into a vertebrate. The particular physiologicalfluid which is tested may have little, if any of the original compound.Therefore, the original presence of the compound might only bedetectable as a metabolite. In many instances, the metabolite may be theglucuronide, either oxy or oxo derivative of the original compound. Inother instances, theoriginal compound may have undergone oxidation,e.g., hydroxylation, reduction, acetylation, deamination, amination,methylation or extensive degradation. Where the metabolite still retainsa substantial portion of the spatial and polar geometry of the originalcompound, it will be frequently possible to make the ligand analog basedon either the original compound or metabolite. Where the metabolite isdistinctively different than the original compound, the ligand analogwill be based on the metabolite.

Of particular interest as metabolites, particularly of the steroids, arethe sulfates and glucuronides.

Besides metabolites of the various drugs, hormones and other compoundspreviously described, of significant interest are metabolites whichrelate to diseased states. illustrative of such compounds are spermine,galactose, phenylpyruvic acid and porphyrin Type 1, which are believedto be diagnostic of certain tumors, galactosemia, phenylketonuria andcongential porphyra, respectively.

Two compounds of interest which are metabolites of epinephrine arevanillylmandelic acid and homovanillic acid. With these compounds,either the hydroxyl and carboxyl groups can be used as the site for X*.

Another general category of interest is the pesticides, e.g.,insecticides, fungicides, bacteriocides and nematocides. Illustrativecompounds include phosphates such as malathion, DDVP, dibrom;carbamates, such as Sevin, etc.

Since many of the biologically active materials are active in only onestereoisomeric form, it is understood that the active form is intendedor the racemate, where the racemate is satisfactory and readilyavailable. The antibodies will be specific for whatever form is used asthe hapten.

Enzymes (A) Enzymes vary widely in their substrates, cofactors,specificity, ubiquitousness, stability to temperature, pH optimum,turnover rate, and the like. Other than inherent factors, there are alsothe practical considerations, that some enzymes have been characterizedextensively, have accurate reproducible assays developed, and arecommercially available. In addition, for the purposes of this invention,the enzymes should either be capable of specific labelling or allow forefficient substitution, so as to be useful in the subject assays. Byspecific labelling is intended selective labelling at a site inrelationship to the active site of the enzyme, so that upon binding ofthe receptor to the ligand, the enzyme is satisfactorily inhibited. Byallowing for efficient substitution to be useful in the subject assay,it is intended that the enzyme be inhibited sufficiently when the ligandis bound to the receptor, and that the degree of substitution requiredto achieve this result does not unreasonably diminish the turnover ratefor the enzyme nor substantially change the enzymes solubilitycharacteristics.

From the standpoint of operability, a very wide variety of enzymes canbe used. But, as a practical matter, there will be a number of groups ofenzymes which are preferred. Employing the International Union ofBiochemists (I.U.B.) classification, the oxidoreductases (1.) and thehydrolases (3.) will be of greatest interest, while the lyases (4.) willbe of lesser interest. Of the we idoreductases, the ones acting on theCHOH group, the aldehyde or keto group, or the C1-lNl-l group as donors(1.1, 1.2, and 1.4 respectively) and those acting on hydrogen peroxideas acceptor (1.1 1) will be preferred. Also, among the oxidoreductasesas preferable will be those which employ nicotinamide adeninedinucleotide, or its phosphate or cytochrome as an acceptor, namely1.X.l and 1. .2, respectively under the 1.U.B. classification. Of thehydrolases, of particular interest are those acting on glycosylcompounds, particularly glycoside hydrolases, and those acting on esterbonds, both organic and inorganic esters, namely the 3.1 and 3.2 groupsrespectively, under the 1.U.B. classification. Other groups of enzymeswhich might find use .are the transferases, the lyases, the isomerases,and the ligases.

In choosing an enzyme for commercialization, as compared to a single orlimited use for scientific investigation, there will be a number ofdesireable criteria. These criteria will be considered below.

The enzyme should be stable when stored for a period of at least 3months, and preferably at least 6 months at temperatures which areconvenient to store in the laboratory, normally 20 C or above.

The enzyme should have a satisfactory turnover rate at or near the pHoptimum for binding to the antibody, this is normally at about pH 6 l0,usually 6.0 to 8.0. Preferably, the enzyme will have the pH optimum forthe turnover rate at or near the pH optimum for binding of the antibodyto the ligand.

A product should be either formed or destroyed as a result of the enzymereaction which absorbs light in the ultra-violet region or the visibleregion, that is in the range of about 250750 nm, preferably 300-600 nm.

Preferably, the enzyme should have a substrate (including cofactors)which has a molecular weight in excess of 300, preferably in excess of500, there being no upper limit. The substrate may either be the naturalsubstrate, or a synthetically available substrate.

Preferably, the enzyme which is employed or other enzymes, with likeactivity, will not be present in the fluid to be measured, or can beeasily removed or deactivated prior to the addition of the assayreagents. Also, one would want that there not be naturally occurringinhibitors for the enzyme present in fluids to be assayed.

Also, although enzymes of up to 600,000 molecular weight can beemployed, usually relatively low molecular weight enzymes will beemployed of from 10,000 to 300,000 molecular weight, more usually fromabout 10,000 to 150,000 molecular weight, and frequently from 10,000 to100,000 molecular weight. Where an enzyme has a plurality of subunitsthe molecular weight limitations refer to the enzyme and not to thesubunits.

For synthetic convenience, it is preferable that there be a reasonablenumber of groups to which the ligand may be bonded, particularly aminogroups. However, other groups to which the ligand may be bonded includehydroxyl groups, thiols, and activated aromatic rings, e.g., phenolic.

Therefore, enzymes will preferably be chosen which are sufficientlycharacterized so as to assure the availability of sites for linking,either in positions which allow for inhibition of the enzyme when theligand is bound to antibody, or there exist a sufficient number ofpositions as to make this occurrence likely.

A list of common enzymes may be found in Hawk, et al., PracticalPhysiological Chemistry, McGraw-Hill Book Company, New York (1954),pages 306 to 307. That list is produced in total as follows, includingthe source of the enzyme, the substrate and the end products.

Name & Class Distribution Substrate End-products HydrolasesCarbohydrases Carbohydrates l. Amylase Pancreas, sal- Starch, dex-Maltose and iva, malt, etc. trin, etc. dextrins 2. Lactase Intestinaljuice Lactose Glucose and and mucosa galactose 3. Maltase Intestinaljuice, Maltose Glucose yeast, etc. 4. Sucrase Intestinal juice Glucoseand yeast, etc. Sucrose fructose 5. Emulsin Plants fi-Cvluco- Glucose,etc.

sides Nucleases Nucleic acid and derivatives l. Polynucleo- Pancreaticjuice Nucleic Nucleotides 'tidase Intestinal juice acid etc. 2.Nucleoti- Intestinal juice Nucleotides Nucleotides and dase and othertissues phosphoric acid 3. Nucleotidase Animal tissues NucleotidesCarbohydrate and bases Amidases Amino compounds and amides l. ArginaseLiver Arginine Ornithine and urea 2. Urease Bacteria, soy- Urea Carbondioxide bean, jack bean and ammonia etc. 3. Glutami- Liver. etc.Glutamine Glutamic acid nase and ammonia 4. Transaminase Animal tissuesGlutamic acid a-Ketogluturic I and oxalacetic acid, aspnrtic acid, etc.acid, etc. Purine Deaminases Purine basesa and derivatives l. AdenaseAnimal tissues Adenine Hypoxanthine and ammonia 2. Guanase Animaltissues Guanine Xanthine and ammonia Peptidases Peptides l.Aminopolypep- Yeast, intestines Polypeptides Simpler peptidase etc.tides and amino acids 2. Carboxypcp- Pancreas Polypeptides Simplerpeptidase tides and amino acids oxidase 3. Peroxidase Enzymes ContainingCoenzymes l and/or ll 1. Alcohol dehydrogenase 2. Malic dehydrogenase 3.lsocritric dehydrogenase 4. Lactic dehydrogenase 5. fl-Hydroxybutyricdehydrogenase 6. Glucose dehydrogenase 7. Robison ester dehydrogenaseGlycerophosphate dehydrogenase 9. Aldehyde dehydrogenase ganisms excepta few species of microorganisms Nearly all plant cells Plant and animaltissues Plant tissues Animal and plant 7 tissues Animal and planttissues Animal and plant tissues gmimal tissues nd yeast Liver, kidneys,and heart Animal tissues Erythrocytes and yeast Animal tissues Livertochromc C in the presence of oxygen A large number of phenols aromaticamines etc.

in the presence of H 0 Various phenolic compounds Ascorbic acid in thepresence of oxygen Ethyl alcohol and other alcohols L( Malic acidL-lsocitric acid Lactic acid acid D-Glucose Robison ester(hexose--phosphate Glycerophosphate Aldehydes Name 8!. ClassDistribution 'Substrate End-products 3. Dipeptidasc Plant and animalDipeptides Amino acids tissues and bacteria 4. Prolinase Animal tissuesProline Proline and a and yeast peptides simpler pep- V v Mm, E@S

Proteinases Proteins l. Pepsin Gastric juice Proteins Proteoses,

peptones, etc. 2. Trypsin Pancreaticjuice Proteins, Polypeptidesproteoses, and amino acid and peptones '3. Cathepsin Animal tissuesProteins Proteoses, V and peptones 4. Rennin Calf stomach CaseinParacasein 5. Chymotrypsin Pancreatic juice Proteins, Polypeptidesproteoses and amino acid and peptones 6. Papain Papaya, other Proteins.

plants proteoses,

and peptones 7. Ficin Fig sap Proteins Proteoses,

"Ji .W Esterases Esters Alcohols and acids l. Lipase Pancreas, castorFats Glycerol and bean, etc. fatty acids 2. Esterases Liver. etc. Ethylbuty- Alcohols and rate, etc. acids 3. Phosphatases Plant and animalEsters of Phosphate and tissues phosphoric alcohol acid 4. SulfatasesAnimal and plant Esters of Sulfuric acid tissues sulfuric and alcoholacid 5. ChOlitleS Blood. tissues Acetylcho- Choline and te rase line Aacetic a cid iron Enzymes l. Catalase All living or- Hydrogen Water andganisms except a peroxide oxygen few species of microorganisms 2.Cytochrome All living or- Reduced cy- Oxidiied cytochrome C and waterOxidation product of substrate and water Oxidation product of substrateDehydroascorbic acid Acetaldehyde and other aldehydes Oxalacetic acidOxalosuccinic acid Pyruvic acid Acetoacetic acid D-Giuconic acidPhosphohexonic acid Phosphogylceric acid W Acids Name & ClassDistribution Substrate End-products Enzymes which Reduce Cytochrome l.Succinic de- Plants, animals Succinic Fumaric acid hydrogenase andmicrooracid (as ordinarily) ganisms prepared) l. Warburgs old YeastReduced co- Oxidized co yellow enzyme enzyme ll enzyme I! and reducedyellow enzyme 2. Diaphorase Bacteria, Reduced co- Oxidized coyeasts,higher enzyme 1 enzyme 1 and plants, and anireduced yelmals lowdiaphorase 3. Haas enzyme Yeast Reduced co- Oxidized coenzyme ll enzymeH and reduced yel- 4. Xanthine oxidase 5. D-amino acid oxidase 6.L-Amino acid oxidases 7. TPN-Cytochrome C reductase 8. DPN Cytochrome Creductase Hydrases l. Fumarase 2. Aconitase 3. Enolase Mutases i.Oiyoxalase Desmolases 1. Zymohexase (aldolase l 2. Carboxylase 3. B-Ketocarboxylases 4. Amino acid decarboxylases 5. Carbonic anhydrase OtherEnzymes l. Phosphorylase 2. Phosphohexoisomerase 3. Hexokinase 4.Phosphoglucomutase Animal tissues Animal tissues Animals, snake venomsYeast, liver Liver, yeast Living oranisms in general Animals and plantsAnimal tissues and yeast Living organisms in general All cells Planttissues Animals, bacteria, plants Plants, animals, bacteria ErythrocytesAnimal and plant tissues Animal and plant tissues Yeast, animal tissuesPlant and animals hypoxanthine xanthine, aldehydes, reduced coenzyme l,etc.

D-Amino Acids 0 L-amino acids Reduced co-;, enzyme ll and cytochrome CReduced c0- enzyme l and cytochrome C Fumaric acid H O Citric acid2-Phosphoglyceric acid Methyl glyoxal and other substituted glyoxalsFructosel .6-diphosphate Pyruvic acid B-Keto acids L-Amino acidsCarbonic acid Starch or glycogen and phosphate Glu c0se-6- phosphateAdenosinetriphosphate Glucose-lphosphate L-Malic acid cis-Aconitic acidand L- isocitric acid Phospyruvic acid H O D Lactic acidDihydroxyacetone phosphoric acid and phosphoglyceric acid Acetaldehydeand C0 a-Keto acids Amines and C0, C0; H O

Glucosel phosphate Fructose-6 phosphate Adenosinediphosphate glucose-6-phosphate Glucose-6- phosphate l. Oxidoreductases 1.1 Acting on theCH-OH group of donors 1.1.1 With NAD or NADP as acceptor l.'alcoholdehydrogenase 6. glycerol dehydrogenase 26. glyoxylate reductase 27.L-lactate dehydrogenase 37. malate dehydrogenase 49. glucose 6-phosphatedehydrogenase 17. mannitol 1-phosphate dehydrogenase 1.1.2 Withcytochrome as an acceptor 3. L-lactate dehydrogenase 1.1.3 With asacceptor 4. glucose oxidase 9. galactose oxidase 1.2 Acting on theCH-Nl-l group of donors 1.4.3 with 0 as acceptor 2. L-amino acid oxidase3. D-amino acid oxidase 1.6 Acting on reduced NAD or NADP as donor1.6.99 With other acceptors diaphorase 1.10 Acting on diphenols andrelated substances as donors 1.10.3 With-O as acceptor l. polyphenoloxidase 3. ascorbate oxidase 1.1 1 Acting on H O as acceptor 1.1 1.1

6. catalase 7. peroxidase 3. Hydrolases 3.1 Acting on ester'bonds 3. l.1 Carboxylic ester hydrolases 7. cholinesterase 3.1.3 Phosphoricmonoester hydrolases l. alkaline phosphatase 3.1.4 Phosphoric diesterhydrolases 3. phospholipase C 3.2 Acting on glycosyl compounds 3.2.1Glycoside hydrolases l. a-amylase 4. cellulase 17. lysozyme 23. Bgalactosiclase 27. amyloglucosidase 31. B-glucuronidase 3.4 Acting onpeptide bonds 3.4.2 Peptidyl-amino acid hydrolase 1. carboxypeptidase A3.4.4 Peptidyl-peptide hydrolase 5. a-chymotrypsin 10. papain 3.5 Actingon C-N bonds other than peptide bonds 3.5.1 in linear amides 5. urease3.6 Acting on acid anhydride bonds 3.6.1 In phosphoryl-containinganhydrides l. inorganic pyrophosphatase 4. Lyases 4.1 Carbon-carbonlyase 4.1.2 Aldehyde lyases 7. aldolase 4.2 Carbon-oxygen lyases 4.2.1Hydrolases l. carbonic anhydrase 4.3 Carbon-nitrogen lyases 4.3.1Ammonia lyases 3. histidase ies will recognize that portion of theligand molecule which extends from the protein, ordinarily the samelinking group will be attached on the same site on the ligand, as wasused in bonding the ligand to the protein to provide the antigenicsubstance.

The functional groups which will be present in the enzyme for linkingare amino (including guanidino), hydroxy, carboxy, and mercapto. Inaddition, activated aromatic groups or imidazole may also serve as asite for linking.

Amino acids having amino groups available for linking include lysine,arginine, and histidine. Amino acids with free hydroxy] groups includeserine, hydroxyproline, tyrosine and threonine. Amino acids which havefree carboxyl groups include aspartic acid and glutamic acid. An aminoacid which has an available mercapto group is cysteine. Finally, theamino acids which have activated aromatic rings are tyrosine andtryptophan.

In most instances, the preferred linking group will be the amino group.However, there will be situations with certain enzymes. where one of theother linking groups will be preferred.

The ligand, of course, will have a great diversity of functionalitieswhich may be present. In addition, as already indicated, thefunctionalities which are present may be modified so as to formadifferent functionality, e.g., keto to hydroxy or an olefin to aldehydeor carboxylic acid. To that extent, the choice of groups for linking tothe ligand may be varied much more widely than the choice of groups forlinking to the enzyme. In both cases, however, a wide variety ofdifferent types of functionalities have been developed, specifically forlinking various compounds to proteins and particularly enzymes.

Where a linking group is employed for bonding the ligand to the enzyme.it will be the more frequent procedure to bond the linking group to theligand to provide an active site for bonding to the enzyme. This may beachieved in a single step or may require a plurality of synthetic steps,including blocking and unblocking the active groups on the ligand, otherthan the one involved in providing the linking group. The linking groupswhich are reported hereafter are solely concerned with the bridgebonding the enzyme and the ligand.

Where a linking group is used, there will normally be from one atom to14 atoms in the chain, more usually from two atoms to eight atoms in thechain bonding the ligand to the enzyme. Where cyclic structures areinvolved, the cyclic structure will be equated to the number of atomsproviding a similar length to the chain.

The linking group (excluding the atoms derived from the ligand andenzyme), when other than a direct bond is involved, will be of fromabout one to 30 atoms carbon, hydrogen, nitrogen, oxygen, phosphorous,and sulfur more usually four to atoms.

Preferably, the linking group will normally be of from zero to 14 carbonatoms, usually from one to eight carbon atoms and from one to eightheteroatoms, and frequently of from one to eight carbon atoms and fromone to four heteroatoms, which are oxygen, sulfur and nitrogen, moreusually oxygen and nitrogen. The most frequent heterofunctionalitiespresent in the linking group are nonoxocarbonyl or thiocarbonyl, amino,l5

imino (oxime or imidate) diazo, or oxy.

A group of linking groups are derived from a group having anonoxocarbonyl functionality and when a second functionality is present,the second functionality may be based on a second nonoxocarbonylfunctionality, a haloalkyl, O-substituted hydroxylamine, imino, amino ordiazo. The linking group will normally have from two to eight carbonatoms and from one to four heteroatoms which are usually oxygen andnitrogen (the heteroatoms of the ligandand enzyme are not included inthe above range of heteroatoms). Such determination is somewhatarbitrary, so that between a carbon atom of the ligand and a carbon atomof the enzyme, there may be as many as six heteroatoms. The heteroatomsmay be part of the linking group chain or branched from the chain, e.g.,nonoxocarbonyl oxygen.

One group of linking groups will have from two to six carbon atoms, moreusually two to four carbon atoms and be an aliphatic non-oxo carbonylfunctionality. Another group of linking groups will have from two toeight carbonatoms and have from one to two heteroatoms, e.g., oxygen andnitrogen, in the chain, such as amino, oximino, diazo, oxy, and thelike.

The following tabulation indicates various linking groups, varying withthe functionalities present on the Ligand Enzyme Ligand Enzyme (onlyprimary amino) ll L S ll .07,

ligand and the enzyme. Except as indicated, the linking group satisfiesone to two valences on the ligand and enzyme functional groups to whichit is bound.

2- bond, hydrocarbylene of from one to IO carbon atoms, morespecifically alkylene of from one to six carbon atoms, alkenylene offrom two to six carbon atoms, alkynylene of from two to six carbonatoms, cycloalkylene of from four to 10 carbon atoms and arylene of fromsix to l0 carbon atoms; oxaalkylene of from four to eight carbon atoms;and azaalkylene of from four to eight carbon atoms;

R alkyl of from one to six carbon atoms;

R hydrogen or alkyl of from one to three carbon atoms;

Z or non-0x0 carbonyl are preferred for bonding to hydroxyl, whilenon-0x0 carbonyl, non-0X0 thiocarbonyl and Z are preferred with amino.

Ligand Enzyme Oxocarbonyl C:O) Amino (-Nll2), hydroxy] will) orinnrt'nplo (S ll).

Z" arylene of from six to 10 carbon atoms. Where the enzyme is to belinked through a carboxyl group of the ligand or a linking group bondedto the ligand, either esters or amides will be prepared. The ligand maybe bonded to any of the linking groups which are appropriate to providea link between the ligand and the alcohol or amine group of the enzymeto form the ester or amide group respectively. When the enzyme has anactivated aromatic ring, the ligand may be bonded to an aromaticdiazonium salt to provide the desired bridge.

1. AN ENZYME-BOUND-LIGAND OF THE FORMULA:
 2. An enzyme-bound-ligand ofthe formula:
 3. An enzyme-bound-ligand according to claim 2, wherein A**is an enzyme of from 10,000 to 300,000 molecular weight and n'' is inthe range of 2 to
 20. 4. An enzyme-bound-ligand according to claim 3,which is an oxidoreductase.
 5. An enzyme-bound-ligand according to claim3, which is a hydrolase.
 6. An enzyme-bound-ligand according to claim 3,which is O3-carboxymethylmorphine conjugate to lysozyme having from 2 to4 O3-carboxymethylmorphine groups.
 7. An enzyme-bound-ligand accordingto claim 3, which is O3-carboxymethylmorphine conjugate to malatedehydrogenase having from 2 to 22 O3-carboxymethylmorphine groups.
 8. Anenzyme-bound-ligand according to claim 3 which isO3-carboxymethylmorphine conjugate to glucose 6-phosphate dehydrogenasehaving from 2 to 22 O3-carboxymethylmorphine groups.
 9. Anenzyme-bound-ligand according to claim 3 which is O3-( Alpha-isopropyl)carboxymethylmorphine conjugate to malate dehydrogenasehaving from 2 to 22 O3-( Alpha -isopropyl)carboxymethylmorphine groups.10. An enzyme-bound-ligand according to claim 3 which is O3-( Alpha-isopropyl)carboxymethylmorphine conjugate to glucose 6-phosphatedehydrogenase having from 2 to 22 O3-( Alpha -isopropyl)carboxymethylmorphine groups.
 11. An enzyme-bound-ligand according to claim 3, whichis O3-imidoylmethylmorphine conjugate to lysozyme, glucose 6-phosphatedehydrogenase or malate dehydrogenase.